Differential Effects of Ethidium Bromide on Cytoplasmic and Mitochondrial Protein Synthesis

February 1975 Volume 50, number 3 FEBS LETTERS

DIFFERENTIAL EFFECTS OF ETHIDIUM BROMIDE ON CYTOPLASMIC AND MITOCHONDRIAL PROTEIN SYNTHESIS

Narayan G. AVADHANI and Robert J. RUTMAN Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania Philadelphia, Pennsylvania, 19174, USA

Received 25 July 1974

1. Introduction seven days in the peritoneal cavity of Swiss colony mice and used for preparing cytoplasmic and mitochon- The phenanthridine dye ethidium bromide (EB) is drial in vitro systems. Mitochondria were prepared by well-known for its ability to bind to DNA molecules. a two step digitonin washing technique and lysed with In eukaryotic cells, even at very low concentrations, nonidet NP40 (Shell Oil Co.) under controlled condi- EB is known to affect mitochondrial specific events [ 13. tions [ 141. 25 000 g supernatant fraction (S-25) In mouse BHK cells 0.1 to 5 gg/ml EB specifically in- from lysed mitochondria contains organelle specific hibits mitochondrial DNA synthesis [2] . This drug is factors and ribosomes highly active in in vitro protein also known to inhibit mitochondrial specific RNA syn- synthesis. Details of preparation of S-25 system from thesis [ 1,3] , probably by binding to the DNA template Ehrlich ascites mitochondria have been described else- thus interfering with chain initiation [4,5]. Consequent- where [ 141 . The cytoplasmic protein synthesizing sys- ly EB has been widely used as a tool to study mitochon- tem was prepared according to Aviv et al. [ 151. Both drial specific transcription [6,7,8] . cytoplasmic and mitochondrial protein synthesis assays Isolated mitochondria from various cell types are were run in 0.05 ml final volumes at 35°C for 45 min. known to incorporate both synthetic and natural poly- The cytoplasmic protein synthesizing system contained nucleotides which participate in intramitochondrial 30 mM Tris-HCl (pH 7.8) 120 mM KCl, 8mM Mg (CH3 translation [9-l 21. Using an in vitro mitochondrial COO)Z, 7 mM 2-mercapto ethanol, 1 mM ATP, 0.25 mM system from Xenopus, Grivell and Metz [ 121 recently GTP, 0.3 mM CTP, 4,5 mM phosphoenol pyruvate, 1.6 demonstrated that EB directly interferes with the pg pyruvate kinase and cytoplasmic extract containing intramitochondrial phenylalanine incorporation di- 230-250 pg protein. The assay mixture for mitochon- rected by imported poly (U). This observation coin- drial protein synthesis contained 30 mM Tris-HCl cides with another indirect observation that EB may (pH 7.5) 120 mM KCl, 15 mM Mg (CHs COO)Z, 7 mM have an inhibitory effect on mitochondrial protein syn- 2-mercaptoethanol, 0.5 mM ATP, 0.1 mM GTP, 0.3 thesis [ 131. We have verified these indirect results using mM CTP, 3 mM phosphoenol pyruvate, 1.6 pg a highly active in vitro protein synthesizing system pyruvate kinase and 20 ~1 of mitochondrial S-25 from Ehrlich ascites mitochondria [ 141. Our results containing 250 pg protein. Both the systems also show that EB specifically inhibits mitochondrial but contained 0.25 OD 260 nm poly (U), 0.5 OD 260 nm not cytoplasmic in vitro protein synthesis by prevent- tRNA (yeast) and 0.5 &i [3H] phenylalanine ing polysome formation. (25 Ci/m mole, New England Nuclear). For both mitochondrial and cytoplasmic systems the above described conditions were found to be optimal for 2. Materials and methods poly (U) directed phenyialanine incorporation. Other details were as described before [14,15]. Ehrlich ascites hypotetraploid cells were grown for

North-Holland Publishing Company - Amsterdam 303 Volume 50. number 3 I;EBS LETTERS I clxu;,ly 197s

3. Results and discussion mitochondrial system with 0.5 /lg EB does not show a polysome-like pattern on a sucrose gradient The direct effects of EB on poly (U) directed phe- (fig. 2B). These results conclusively show that EB nylalanine incorporation in mitochondrial and cyto- specifically inhibits mitochondrial protein synthesis plasmic in vitro systems are shown in fig. 1. At the high- without any detectable effect on the cytoplasmic sys- est amount of EB used. i.e., 10 pg, the cytoplasmic system in vitro. tem is inhibited by only about 10%. In contrast, EB has Although these experiments show that the inhibitory a pronounced inhibitory effect on the mitochondrial effect of EB on the mitochondrial system is probably system and even at the 1 yg level, protein synthesis is due to interference with some stage of the initiation inhibited by about 60%. Considering the very low con- process thereby preventing polysome formation, the centrations of EB needed to inhibit mitochondrial DNA precise effect is not yet known. Since it is known that and RNA synthesis, the concentrations of EB needed EB has an affinity fo double-stranded RNA, it is possi- to affect the protein synthesis in the present experiments ble that it binds to mRNA and prevents the initiation of are high. However, similar effects have been noted by protein synthesis. However, the fact that there is no others [4,5]. EB inhibition of poly (U) directed cytoplasmic pro- In order to further examine the inhibitory effects of tein synthesis suggests that inhibition of the mitochon- EB on the mitochondrial and cytoplasmic systems ex- drial system is not due to the binding of the drug to periments were carried out using 3H-labeled poly (U) mRNA but may involve binding of the drug to mito- and the reaction mixture was analyzed. As seen in fig. chondrial ribosomes. The exact mechanism of action 2A, within 7-8 min of the reaction in both mitochon- will have to be tested using radio-labeled EB and natural drial and cytoplasmic systems counts are seen in poly- mRNA’s somal region. The cytoplasmic system containing Several studies in the past showing either the inhib- 0.5 I-18EB also shows a typical polysomal pattern itory effects of EB on amino acid incorporation by isola (fig. 2B) comparable to that seen in the control ex- ted mitochondria [16,17] or reduced amounts of mi- periment without EB (see fig. 2A). In contrast, the tochondrial polysomes or polysomal RNA contents

100

,0--c ??

B. 60 c .-> 2 40

s 20

0.8 I 5 8 IO EB Added (pghbe)

Fig. 1. Effect of EB on mitochondrial (o--o--o), and cytoplasmic (.-@A) protein synthesis. Details of protein synthesis assays were as in Materials and methods. Amino acid incorporation activity in tubes with 0.1 pug EB were considered to be 100%. Actual CPM at this concentration were 225 000 CPM for cytoplasmic and 189 000 for mitochondrial systems. Tubes without EB gave 226 000 CPM for cytoplasmic and 205 000 CPM for mitochondrial systems.

304 Volume 50, number 3 FEBS LETTERS February 1975

80 S In conclusion, our data confirm and extend the ob- servations of Grivell and Metz [ 121 and Lederman and Attardi [ 131 that EB inhibits mitochondrial specific protein synthesis.

Acknowledgements

This research was supported in part by a USPHS Grant CA-05295.

References

[l] Penman, S., Fan, H., Perlman, S., Rosebash, M., Weinber, R., and Zylber, S. (1970) Cold Spring Harbor Sympos. Quant. Biol., 35,561. [2] Nass, M. M. K. (1972) Exptl. Cell Res., 72, 211. [3] Attardi, G., Aloni, Y., Attardi, B., Ojala, D., Pica-Mattocia, L., Robberson, D. L., and Storrie, B. (1970) Cold Spring I Harbor Symp. Quant. Biol., 35,599. M [4] Richardson, J. P. and Parker, S. R., (1973) J. Mol. Biol., b.*.* +EB 78, 715. ‘.N.. [5] Richardson, J. P. (1973) J. Mol. Biol., 78, 703. I ??&r. I 0 [61 Perlman, S., Abelson, H. T., and Penman, S. (1973) Proc. IO 20 30 Natl. Acad. Sci. U.S. 70, 350. [71 Mahler, H. R. and Dawidowicz, K. (1973) Proc. Natl. TOP FRACTIONS BOTTOM Acad. Sci., U.S., 70, 111. I81 Avadhani, N. G., Battula, N. and Rutman, R. J. (1973) Fig. 2. Effect of EB on polysome formation in mitochondrial Biochemistry, 12,4 122. (*A-+) and cytoplasmic (o--o--i)) systems. The reaction PI Swanson, R. F. (197 1) Nature, 23 1, 3 1. was run as described in Materials and methods, except that 2 pg [lOI Kisselev, 0. I. and Gaitskhoki, V. S. (1972) Biokhimia, (7.0 X 10 CPM) “H-labeled poly (U) (Miles Laboratories) were 37, 1224. added. After 7.5 min reaction, aliquots containing 5-6000 [Ill Kyriakidis, D. and Georgatos, J. G. (1973) Biochem. CPM were layered on 4.5 ml of lo-36% linear sucrose gradients Biophys. Res. Commun., 53, 1167. and centrifuged at 145 000 g for 2 hr. Gradients were frac- [I21 Grivell, L. A. and Metz, V. (1973) Biochem. Biophys. tionated and 4 drop fractions were counted with 10 ml cab-o- Res. Commun., 55, 125. sil mixture. Fig. A represent control mitochondrial and cyto-’ I131 Lederman, M. and Attardi, G. (1973) .I. Mol. Biol., plasmic systems without EB. The dashed line shows the sedi- 78, 275. mentation profile of poly (U) alone. Fig. B shows the sedimen- [I41 Avadhani, N. G. and Rutman, R. J. (1974) Biochem. tation profdes of mitochondrial and cytoplasmic systems with Biophys. Res. Commun., 58,42. 0.5 pg EB. Cytoplasmic ribosomes were used as markers. [I51 Aviv, H., Biome, I. and Leder, P. (1971) Proc. Natl. Acad. Sci., U.S., 68, 2303. [I61 Schatz, G., Groot, G. S. P., Mason, T., Rouslin, W., [6,7] have been interpreted in favor of the hypothesis Wharton, D. C., and Slatzgaber, J. (1972) Fed. Proc. 31,21. that mitochondria are autonomous with respect to [17] Kroon, A. M. and De Vries, H. (1971) in ‘Autonomy their mRNA synthesis. However, because of the direct and Biogenesis of Mitochondria and Chloroplasts’ effect of the drug on mitochondrial specific protein (Boardman, Linnane and Smillie, eds.) p. 318, North synthesis, these arguments will have to be re-evaluated. Holland, Amsterdam.

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